The Gibbs free energy difference between native and unfolded states ("stability") is one of the fundamental characteristics of a protein. By exploiting the thermodynamic linkage between ligand binding and stability, interactions of a protein with small molecules, nucleic acids, or other proteins can be detected and quantified. Determination of protein stability can therefore provide a universal monitor of biochemical function. Yet, the use of stability measurements as a functional probe is underutilized, because such experiments traditionally require large amounts of protein and special instrumentation. Here we present the quantitative cysteine reactivity (QCR) technique to determine protein stabilities rapidly and accurately using only picomole quantities of material and readily accessible laboratory equipment. We demonstrate that QCR-derived stabilities can be used to measure ligand binding over a wide range of ligand concentrations and affinities. We anticipate that this technique will have broad applications in high-throughput protein engineering experiments and functional genomics. Study holds ProTherm entries: 25489, 25490, 25491, 25492, 25493, 25494, 25495, 25496, 25497, 25498 Extra Details: conformational stability; thermal stability; ligand-binding affinity; linkage analysis; thiol protection
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:55 p.m.
|Number of data points||18|
|Proteins||Thermonuclease ; Thermonuclease ; Ribose import binding protein RbsB ; Ribose import binding protein RbsB|
|Assays/Quantities/Protocols||Experimental Assay: dHvH temp:25.0 C ; Experimental Assay: dG temp:25.0 C ; Experimental Assay: dHvH temp:20.0 C ; Experimental Assay: dG temp:20.0 C ; Experimental Assay: Tm ; Derived Quantity: dTm|
|Libraries||Mutations for sequence ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ ; Mutations for sequence KDTIALVVSTLNNPFFVSLKDGAQKEADKLGYNLVVLDSQNNPAKELANVQDLTVRGTKILLINPTDSDAVGNAVKMANQANIPVITLDRQATKGEVVSHIASDNVLGGKIAGDYIAKKAGEGAKVIELQGIAGTSAARERGEGFQQAVAAHKFNVLASQPADFDRIKGLNVMQNLLTAHPDVQAVFAQNDEMALGALRALQTAGKSDVMVVGFDGTPDGEKAVNDGKLAATIAQLPDQIGAKGVETADKVLKGEKVQAKYPVDLKLVVKQ|